Uracyl spirooxetane nucleosides

ABSTRACT

The present invention also relates to processes for preparing said compounds, pharmaceutical compositions containing them and their use, alone or in combination with other HCV inhibitors, in HCV therapy.

BACKGROUND OF THE INVENTION

This invention relates to spirooxetane nucleosides and nucleotides thatare inhibitors of the hepatitis C virus (HCV).

HCV is a single stranded, positive-sense RNA virus belonging to theFlaviviridae family of viruses in the hepacivirus genus. The NS5B regionof the RNA polygene encodes a RNA dependent RNA polymerase (RdRp), whichis essential to viral replication. Following the initial acuteinfection, a majority of infected individuals develop chronic hepatitisbecause HCV replicates preferentially in hepatocytes but is not directlycytopathic. In particular, the lack of a vigorous T-lymphocyte responseand the high propensity of the virus to mutate appear to promote a highrate of chronic infection. Chronic hepatitis can progress to liverfibrosis, leading to cirrhosis, end-stage liver disease, and HCC(hepatocellular carcinoma), making it the leading cause of livertransplantations. There are six major HCV genotypes and more than 50subtypes, which are differently distributed geographically. HCV genotype1 is the predominant genotype in Europe and in the US. The extensivegenetic heterogeneity of HCV has important diagnostic and clinicalimplications, perhaps explaining difficulties in vaccine development andthe lack of response to current therapy.

Transmission of HCV can occur through contact with contaminated blood orblood products, for example following blood transfusion or intravenousdrug use. The introduction of diagnostic tests used in blood screeninghas led to a downward trend in post-transfusion HCV incidence. However,given the slow progression to the end-stage liver disease, the existinginfections will continue to present a serious medical and economicburden for decades.

Current HCV therapy is based on (pegylated) interferon-alpha (IFN-α) incombination with ribavirin. This combination therapy yields a sustainedvirologic response in more than 40% of patients infected by genotype 1HCV and about 80% of those infected by genotypes 2 and 3. Beside thelimited efficacy against HCV genotype 1, this combination therapy hassignificant side effects and is poorly tolerated in many patients. Majorside effects include influenza-like symptoms, hematologic abnormalities,and neuropsychiatric symptoms. Hence there is a need for more effective,convenient and better-tolerated treatments.

Recently, therapy possibilities have extended towards the combination ofa HCV protease inhibitor (e.g. Telaprevir or boceprevir) and (pegylated)interferon-alpha (IFN-α)/ribavirin.

Experience with HIV drugs, in particular with HIV protease inhibitors,has taught that sub-optimal pharmacokinetics and complex dosing regimesquickly result in inadvertent compliance failures. This in turn meansthat the 24 hour trough concentration (minimum plasma concentration) forthe respective drugs in an HIV regime frequently falls below the IC₉₀ orED₉₀ threshold for large parts of the day. It is considered that a 24hour trough level of at least the IC₅₀, and more realistically, the IC₉₀or ED₉₀, is essential to slow down the development of drug escapemutants. Achieving the necessary pharmacokinetics and drug metabolism toallow such trough levels provides a stringent challenge to drug design.

The NS5B RdRp is essential for replication of the single-stranded,positive sense, HCV RNA genome. This enzyme has elicited significantinterest among medicinal chemists. Both nucleoside and non-nucleosideinhibitors of NS5B are known. Nucleoside inhibitors can act as a chainterminator or as a competitive inhibitor, or as both. In order to beactive, nucleoside inhibitors have to be taken up by the cell andconverted in vivo to a triphosphate. This conversion to the triphosphateis commonly mediated by cellular kinases, which imparts additionalstructural requirements on a potential nucleoside polymerase inhibitor.In addition this limits the direct evaluation of nucleosides asinhibitors of HCV replication to cell-based assays capable of in situphosphorylation.

Several attempts have been made to develop nucleosides as inhibitors ofHCV RdRp, but while a handful of compounds have progressed into clinicaldevelopment, none have proceeded to registration. Amongst the problemswhich HCV-targeted nucleosides have encountered to date are toxicity,mutagenicity, lack of selectivity, poor efficacy, poor bioavailability,sub-optimal dosage regimes and ensuing high pill burden and cost ofgoods.

Spirooxetane nucleosides, in particular1-(8-hydroxy-7-(hydroxy-methyl)-1,6-dioxaspiro[3.4]octan-5-yl)pyrimidine-2,4-dionederivatives and their use as HCV inhibitors are known fromWO2010/130726, and WO2012/062869, including CAS-1375074-52-4.

There is a need for HCV inhibitors that may overcome at least one of thedisadvantages of current HCV therapy such as side effects, limitedefficacy, the emerging of resistance, and compliance failures, orimprove the sustained viral response.

The present invention concerns a group of HCV-inhibiting uracylspirooxetane derivatives with useful properties regarding one or more ofthe following parameters: antiviral efficacy towards at least one of thefollowing genotypes 1a, 1b, 2a, 2b, 3,4 and 6, favorable profile ofresistance development, lack of toxicity and genotoxicity, favorablepharmacokinetics and pharmacodynamics and ease of formulation andadministration.

DESCRIPTION OF THE INVENTION

In one aspect the present invention provides compounds that can berepresented by the formula I:

including any possible stereoisomer thereof, wherein:R⁹ is C₁-C₆alkyl, phenyl, C₃-C₇cycloalkyl or C₁-C₃alkyl substituted with1, 2 or 3 substituents each independently selected from phenyl, naphtyl,C₃-C₆cycloalkyl, hydroxy, or C₁-C₆alkoxy;or a pharmaceutically acceptable salt or solvate thereof.

Of particular interest are compounds of formula I or subgroups thereofas defined herein, that have a structure according to formula Ia:

In one embodiment of the present invention, R⁹ is C₁-C₆alkyl, phenyl,C₃-C₇cycloalkyl or C₁-C₃alkyl substituted with 1 substituent selectedfrom phenyl, C₃-C₆cycloalkyl, hydroxy, or C₁-C₆alkoxy. In anotherembodiment of the present invention, R⁹ in Formula I or Ia is C₁-C₆alkylor C₁-C₂alkyl substituted with phenyl C₁-C₂alkoxy or C₃-C₆cycloalkyl. Ina more preferred embodiment, R⁹ is C₂-C₄alkyl and in a most preferredembodiment, R⁹ is i-propyl.

A preferred embodiment according to the invention is a compoundaccording to formula Ib:

including any pharmaceutically acceptable salt or solvate thereof andthe use of compound (V) in the synthesis of a compound according toFormula I, Ia or Ib.

The invention further relates to a compound of formula V:

including any pharmaceutically acceptable salt or solvate thereof andthe use of compound (V) in the synthesis of a compound according toFormula I, Ia or Ib.

In addition, the invention relates to a compound of formula VI:

including any stereochemical form and/or pharmaceutically acceptablesalt or solvate thereof.

Additionally, the invention relates to a pharmaceutical compositioncomprising a compound according to Formula I, Ia or Ib, and apharmaceutically acceptable carrier. The invention also relates to aproduct containing (a) a compound of formula I, Ia or Ib a, and (b)another HCV inhibitor, as a combined preparation for simultaneous,separate or sequential use in the treatment of HCV infections

Yet another aspect of the invention relates to a compound according toFormula I, Ia or Ib or a pharmaceutical composition according to thepresent invention for use as a medicament, preferably for use in theprevention or treatment of an HCV infection in a mammal.

In a further aspect, the invention provides a compound of formula I Iaor Ib or a pharmaceutically acceptable salt, hydrate, or solvatethereof, for use in the treatment or prophylaxis (or the manufacture ofa medicament for the treatment or prophylaxis) of HCV infection.Representative HCV genotypes in the context of treatment or prophylaxisin accordance with the invention include genotype 1b (prevalent inEurope) or 1a (prevalent in North America). The invention also providesa method for the treatment or prophylaxis of HCV infection, inparticular of the genotype 1a or 1b.

Of particular interest is compound 8a mentioned in the section“Examples” as well as the pharmaceutically acceptable acid additionsalts of this compound.

The compounds of formula I have several centers of chirality, inparticular at the carbon atoms 1′, 2′, 3′, and 4′. Although thestereochemistry at these carbon atoms is fixed, the compounds maydisplay at least 75%, preferably at least 90%, such as in excess of 95%,or of 98%, enantiomeric purity at each of the chiral centers.

The phosphorus center can be present as R_(P) or S_(P), or a mixture ofsuch stereoisomers, including racemates. Diastereoisomers resulting fromthe chiral phosphorus center and a chiral carbon atom may exist as well.

The compounds of formula I are represented as a defined stereoisomer,except for the stereoisomerism at the phosphorous atom. The absoluteconfiguration of such compounds can be determined using art-knownmethods such as, for example, X-ray diffraction or NMR and/orimplication from starting materials of known stereochemistry.Pharmaceutical compositions in accordance with the invention willpreferably comprise stereoisomerically pure forms of the indicatedstereoisomer of the particular compound of formula I.

Pure stereoisomeric forms of the compounds and intermediates asmentioned herein are defined as isomers substantially free of otherenantiomeric or diastereomeric forms of the same basic molecularstructure of said compounds or intermediates. In particular, the term“stereoisomerically pure” concerns compounds or intermediates having astereoisomeric excess of at least 80% (i.e. minimum 90% of one isomerand maximum 10% of the other possible isomers) up to a stereoisomericexcess of 100% (i.e. 100% of one isomer and none of the other), more inparticular, compounds or intermediates having a stereoisomeric excess of90% up to 100%, even more in particular having a stereoisomeric excessof 94% up to 100% and most in particular having a stereoisomeric excessof 97% up to 100%, or of 98% up to 100%. The terms “enantiomericallypure” and “diastereomerically pure” should be understood in a similarway, but then having regard to the enantiomeric excess, and thediastereomeric excess, respectively, of the mixture in question.

Pure stereoisomeric forms of the compounds and intermediates of thisinvention may be obtained by the application of art-known procedures.For instance, enantiomers may be separated from each other by theselective crystallization of their diastereomeric salts with opticallyactive acids or bases. Examples thereof are tartaric acid,dibenzoyl-tartaric acid, ditoluoyltartaric acid and camphorsulfonicacid. Alternatively, enantiomers may be separated by chromatographictechniques using chiral stationary layers. Said pure stereochemicallyisomeric forms may also be derived from the corresponding purestereochemically isomeric forms of the appropriate starting materials,provided that the reaction occurs stereospecifically. Preferably, if aspecific stereoisomer is desired, said compound is synthesized bystereospecific methods of preparation. These methods will advantageouslyemploy enantiomerically pure starting materials.

The diastereomeric racemates of the compounds of formula I can beobtained separately by conventional methods. Appropriate physicalseparation methods that may advantageously be employed are, for example,selective crystallization and chromatography, e.g. columnchromatography.

The pharmaceutically acceptable addition salts comprise thetherapeutically active non-toxic acid and base addition salt forms ofthe compounds of formula I. Of interest are the free, i.e. non-saltforms of the compounds of formula I, or of any subgroup of compounds offormula I specified herein.

The pharmaceutically acceptable acid addition salts can conveniently beobtained by treating the base form with such appropriate acid.Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propionic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxyl-butanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, palmoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds of formula I containing an acidic proton may also beconverted into their non-toxic metal or amine addition salt forms bytreatment with appropriate organic and inorganic bases. Appropriate basesalt forms comprise, for example, the ammonium salts, the alkali andearth alkaline metal salts, e.g. the lithium, sodium, potassium,magnesium, calcium salts and the like, salts with organic bases, e.g.the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts withamino acids such as, for example, arginine, lysine and the like.

The term “solvates” covers any pharmaceutically acceptable solvates thatthe compounds of formula I as well as the salts thereof, are able toform. Such solvates are for example hydrates, alcoholates, e.g.ethanolates, propanolates, and the like.

Some of the compounds of formula I may also exist in their tautomericform. For example, tautomeric forms of amide (—C(═O)—NH—) groups areiminoalcohols (—C(OH)═N—), which can become stabilized in rings witharomatic character. The uridine base is an example of such a form. Suchforms, although not explicitly indicated in the structural formulaerepresented herein, are intended to be included within the scope of thepresent invention.

SHORT DESCRIPTION OF THE FIGURE

FIG. 1: In vivo efficacy of compound 8a and CAS-1375074-52-4 asdetermined in a humanized hepatocyte mouse model.

DEFINITIONS

As used herein “C₁-C_(n)alkyl” as a group or part of a group definessaturated straight or branched chain hydrocarbon radicals having from 1to n carbon atoms. Accordingly, “C₁-C₄alkyl” as a group or part of agroup defines saturated straight or branched chain hydrocarbon radicalshaving from 1 to 4 carbon atoms such as for example methyl, ethyl,1-propyl, 2-propyl, 1-butyl, 2-butyl, 2-methyl-1-propyl,2-methyl-2-propyl. “C₁-C₆alkyl” encompasses C₁-C₄alkyl radicals and thehigher homologues thereof having 5 or 6 carbon atoms such as, forexample, 1-pentyl, 2-pentyl, 3-pentyl, 1-hexyl, 2-hexyl,2-methyl-1-butyl, 2-methyl-1-pentyl, 2-ethyl-1-butyl, 3-methyl-2-pentyl,and the like. Of interest amongst C₁-C₆alkyl is C₁-C₄alkyl.

‘C₁-C_(n)alkoxy’ means a radical —O—C₁-C_(n)alkyl wherein C₁-C_(n)alkylis as defined above. Accordingly, ‘C₁-C₆alkoxy’ means a radical—O—C₁-C₆alkyl wherein C₁-C₆alkyl is as defined above. Examples ofC₁-C₆alkoxy are methoxy, ethoxy, n-propoxy, or isopropoxy. Of interestis ‘C₁-C₂alkoxy’, encompassing methoxy and ethoxy.

“C₃-C₆cycloalkyl” includes cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl.

In one embodiment, the term “phenyl-C₁-C₆alkyl” is benzyl.

As used herein, the term ‘(═O)’ or ‘oxo’ forms a carbonyl moiety whenattached to a carbon atom. It should be noted that an atom can only besubstituted with an oxo group when the valency of that atom so permits.

The term “monophosphate, diphosphate or triphosphate ester” refers togroups:

Where the position of a radical on a molecular moiety is not specified(for example a substituent on phenyl) or is represented by a floatingbond, such radical may be positioned on any atom of such a moiety, aslong as the resulting structure is chemically stable. When any variableis present more than once in the molecule, each definition isindependent.

Whenever used herein, the term ‘compounds of formula I’, or ‘the presentcompounds’ or similar terms, it is meant to include the compounds ofFormula I, Ia and Ib, including the possible stereochemically isomericforms, and their pharmaceutically acceptable salts and solvates.

The present invention also includes isotope-labeled compounds of formulaI or any subgroup of formula I, wherein one or more of the atoms isreplaced by an isotope that differs from the one(s) typically found innature. Examples of such isotopes include isotopes of hydrogen, such as²H and ³H; carbon, such as ¹¹C, ¹³C and ¹⁴C; nitrogen, such as ¹³N and¹⁵N; oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O; phosphorus, such as ³¹P and ³²P,sulphur, such as ³⁵S; fluorine, such as ¹⁸F; chlorine, such as ³⁶Cl;bromine such as ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br; and iodine, such as ¹²³I,¹²⁴I, ¹²⁵I and ¹³¹I. Isotope-labeled compounds of the invention can beprepared by processes analogous to those described herein by using theappropriate isotope-labeled reagents or starting materials, or byart-known techniques. The choice of the isotope included in anisotope-labeled compound depends on the specific application of thatcompound. For example, for tissue distribution assays, a radioactiveisotope such as ³H or ¹⁴C is incorporated. For radio-imagingapplications, a positron emitting isotope such as ¹¹C, ¹⁸F, ¹³N or ¹⁵Owill be useful. The incorporation of deuterium may provide greatermetabolic stability, resulting in, e.g. an increased in vivo half lifeof the compound or reduced dosage requirements.

General Synthetic Procedures

The following schemes are just meant to be illustrative and are by nomeans limiting the scope.

The starting material1-[(4R,5R,7R,8R)-8-hydroxy-7-(hydroxymethyl)-1,6-dioxaspiro[3.4]octan-5-yl]pyrimidine-2,4(1H,3H)-dione(1) can be prepared as exemplified in WO2010/130726. Compound (1) isconverted into compounds of the present invention via a p-methoxybenzylprotected derivative (4) as exemplified in the following Scheme 1.

In Scheme 1, R⁹ can be C₁-C₆alkyl, phenyl, naphtyl, C₃-C₇cycloalkyl orC₁-C₃alkyl substituted with 1,2 or 3 substituents each independentlyselected from phenyl, C₃-C₆cycloalkyl, hydroxy, or C₁-C₆alkoxy,preferably R⁹ is C₁-C₆alkyl or C₁-C₂alkyl substituted with phenyl,C₁-C₂alkoxy or C₃-C₆cycloalkyl, even more preferably R⁹ is C₂-C₄alkyland most preferably R⁹ is i-propyl.

In a further aspect, the present invention concerns a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof formula I as specified herein, and a pharmaceutically acceptablecarrier. Said composition may contain from 1% to 50%, or from 10% to 40%of a compound of formula I and the remainder of the composition is thesaid carrier. A therapeutically effective amount in this context is anamount sufficient to act in a prophylactic way against HCV infection, toinhibit HCV, to stabilize or to reduce HCV infection, in infectedsubjects or subjects being at risk of becoming infected. In still afurther aspect, this invention relates to a process of preparing apharmaceutical composition as specified herein, which comprisesintimately mixing a pharmaceutically acceptable carrier with atherapeutically effective amount of a compound of formula I, asspecified herein.

The compounds of formula I or of any subgroup thereof may be formulatedinto various pharmaceutical forms for administration purposes. Asappropriate compositions there may be cited all compositions usuallyemployed for systemically administering drugs. To prepare thepharmaceutical compositions of this invention, an effective amount ofthe particular compound, optionally in addition salt form or metalcomplex, as the active ingredient is combined in intimate admixture witha pharmaceutically acceptable carrier, which carrier may take a widevariety of forms depending on the form of preparation desired foradministration. These pharmaceutical compositions are desirable inunitary dosage form suitable, particularly, for administration orally,rectally, percutaneously, or by parenteral injection. For example, inpreparing the compositions in oral dosage form, any of the usualpharmaceutical media may be employed such as, for example, water,glycols, oils, alcohols and the like in the case of oral liquidpreparations such as suspensions, syrups, elixirs, emulsions andsolutions; or solid carriers such as starches, sugars, kaolin,lubricants, binders, disintegrating agents and the like in the case ofpowders, pills, capsules, and tablets. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage unit forms, in which case solid pharmaceutical carriers areobviously employed. For parenteral compositions, the carrier willusually comprise sterile water, at least in large part, though otheringredients, for example, to aid solubility, may be included. Injectablesolutions, for example, may be prepared in which the carrier comprisessaline solution, glucose solution or a mixture of saline and glucosesolution. Injectable suspensions may also be prepared in which caseappropriate liquid carriers, suspending agents and the like may beemployed. Also included are solid form preparations intended to beconverted, shortly before use, to liquid form preparations. In thecompositions suitable for percutaneous administration, the carrieroptionally comprises a penetration enhancing agent and/or a suitablewetting agent, optionally combined with suitable additives of any naturein minor proportions, which additives do not introduce a significantdeleterious effect on the skin. The compounds of the present inventionmay also be administered via oral inhalation or insufflation in the formof a solution, a suspension or a dry powder using any art-known deliverysystem.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in unit dosage form for ease ofadministration and uniformity of dosage. Unit dosage form as used hereinrefers to physically discrete units suitable as unitary dosages, eachunit containing a predetermined quantity of active ingredient calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. Examples of such unit dosage forms aretablets (including scored or coated tablets), capsules, pills,suppositories, powder packets, wafers, injectable solutions orsuspensions and the like, and segregated multiples thereof.

The compounds of formula I show activity against HCV and can be used inthe treatment and/or prophylaxis of HCV infection or diseases associatedwith HCV. The latter include progressive liver fibrosis, inflammationand necrosis leading to cirrhosis, end-stage liver disease, and HCC. Thecompounds of this invention moreover are believed to be active againstmutated strains of HCV and show a favorable pharmacokinetic profile andhave attractive properties in terms of bioavailability, including anacceptable half-life, AUC (area under the curve) and peak values andlacking unfavorable phenomena such as insufficient quick onset andtissue retention.

The in vitro antiviral activity against HCV of the compounds of formulaI can be tested in a cellular HCV replicon system based on Lohmann etal. (1999) Science 285:110-113, with the further modifications describedby Krieger et al. (2001) Journal of Virology 75: 4614-4624 (incorporatedherein by reference), which is further exemplified in the examplessection. This model, while not a complete infection model for HCV, iswidely accepted as the most robust and efficient model of autonomous HCVRNA replication currently available. It will be appreciated that it isimportant to distinguish between compounds that specifically interferewith HCV functions from those that exert cytotoxic or cytostatic effectsin the HCV replicon model, and as a consequence cause a decrease in HCVRNA or linked reporter enzyme concentration. Assays are known in thefield for the evaluation of cellular cytotoxicity based for example onthe activity of mitochondrial enzymes using fluorogenic redox dyes suchas resazurin. Furthermore, cellular counter screens exist for theevaluation of non-selective inhibition of linked reporter gene activity,such as firefly luciferase. Appropriate cell types can be equipped bystable transfection with a luciferase reporter gene whose expression isdependent on a constitutively active gene promoter, and such cells canbe used as a counter-screen to eliminate non-selective inhibitors.

Due to their anti-HCV properties, the compounds of formula I, includingany possible stereoisomers, the pharmaceutically acceptable additionsalts or solvates thereof, are useful in the treatment of warm-bloodedanimals, in particular humans, infected with HCV, and in the prophylaxisof HCV infections. The compounds of the present invention may thereforebe used as a medicine, in particular as an anti-HCV or a HCV-inhibitingmedicine. The present invention also relates to the use of the presentcompounds in the manufacture of a medicament for the treatment or theprevention of HCV infection. In a further aspect, the present inventionrelates to a method of treating a warm-blooded animal, in particularhuman, infected by HCV, or being at risk of becoming infected by HCV,said method comprising the administration of an anti-HCV effectiveamount of a compound of formula I, as specified herein. Said use as amedicine or method of treatment comprises the systemic administration toHCV-infected subjects or to subjects susceptible to HCV infection of anamount effective to combat the conditions associated with HCV infection.

In general it is contemplated that an antiviral effective daily amountwould be from about 1 to about 30 mg/kg, or about 2 to about 25 mg/kg,or about 5 to about 15 mg/kg, or about 8 to about 12 mg/kg body weight.Average daily doses can be obtained by multiplying these daily amountsby about 70. It may be appropriate to administer the required dose astwo, three, four or more sub-doses at appropriate intervals throughoutthe day. Said sub-doses may be formulated as unit dosage forms, forexample, containing about 1 to about 2000 mg, or about 50 to about 1500mg, or about 100 to about 1000 mg, or about 150 to about 600 mg, orabout 100 to about 400 mg of active ingredient per unit dosage form.

As used herein the term “about” has the meaning known to the personskilled in the art. In certain embodiments the term “about” may be leftout and the exact amount is meant. In other embodiments the term “about”means that the numerical following the term “about” is in the range of±15%, or of ±10%, or of ±5%, or of ±1%, of said numerical value.

Examples

Synthesis of Compound (2)

Compound (2) can be prepared by dissolving compound (1) in pyridine andadding 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane. The reaction isstirred at room temperature until complete. The solvent is removed andthe product redissolved in CH₂Cl₂ and washed with saturated NaHCO₃solution. Drying on MgSO₄ and removal of the solvent gives compound (2).

Synthesis of Compound (3)

Compound (3) is prepared by reacting compound (2) withp-methoxybenzylchloride in the presence of DBU as the base in CH₃CN.

Synthesis of Compound (4)

Compound (4) is prepared by cleavage of the bis-silyl protecting groupin compound (3) using TBAF as the fluoride source.

Synthesis of Compound (6a)

A solution of isopropyl alcohol (3.86 mL, 0.05 mol) and triethylamine(6.983 mL, 0.05 mol) in dichloromethane (50 mL) was added to a stirredsolution of POCl₃ (5) (5.0 mL, 0.0551 mol) in DCM (50 mL) dropwise overa period of 25 min at −5° C. After the mixture stirred for 1 h, thesolvent was evaporated, and the residue was suspended in ether (100 mL).The triethylamine hydrochloride salt was filtered and washed with ether(20 mL). The filtrate was concentrated, and the residue was distilled togive the (6) as a colorless liquid (6.1 g, 69% yield).

Synthesis of Compound (7a)

To a stirred suspension of (4) (2.0 g, 5.13 mmol) in dichloromethane (50mL) was added triethylamine (2.07 g, 20.46 mmol) at room temperature.The reaction mixture was cooled to −20° C., and then (6a) (1.2 g, 6.78mmol) was added dropwise over a period of 10 min. The mixture wasstirred at this temperature for 15 min and then NMI was added (0.84 g,10.23 mmol), dropwise over a period of 15 min. The mixture was stirredat −15° C. for 1 h and then slowly warmed to room temperature in 20 h.The solvent was evaporated, the mixture was concentrated and purified bycolumn chromatography using petroleum ether/EtOAc (10:1 to 5:1 as agradient) to give (7a) as white solid (0.8 g, 32% yield).

Synthesis of Compound (8a)

To a solution of (7a) in CH₃CN (30 mL) and H₂O (7 mL) was add CANportion wise below 20° C. The mixture was stirred at 15-20° C. for 5 hunder N₂. Na₂SO₃ (370 mL) was added dropwise into the reaction mixturebelow 15° C., and then Na₂CO₃ (370 mL) was added. The mixture wasfiltered and the filtrate was extracted with CH₂Cl₂ (100 mL*3). Theorganic layer was dried and concentrated to give the residue. Theresidue was purified by column chromatography to give the targetcompound (8a) as white solid. (Yield: 55%)

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.45 (dd, J=7.53, 6.27 Hz, 6H),2.65-2.84 (m, 2H), 3.98 (td, J=10.29, 4.77 Hz, 1H), 4.27 (t, J=9.66 Hz,1H), 4.43 (ddd, J=8.91, 5.77, 5.65 Hz, 1H), 4.49-4.61 (m, 1H), 4.65 (td,J=7.78, 5.77 Hz, 1H), 4.73 (d, J=7.78 Hz, 1H), 4.87 (dq, J=12.74, 6.30Hz, 1H), 5.55 (br. s., 1H), 5.82 (d, J=8.03 Hz, 1H), 7.20 (d, J=8.03 Hz,1H), 8.78 (br. s., 1H); ³¹P NMR (CHLOROFORM-d) δ ppm −7.13; LC-MS: 375(M+1)+

Step 1: Synthesis of Compound (9)

Compound (1), CAS 1255860-33-3 (1200 mg, 4.33 mmol) and1,8-bis(dimethyl-aminonaphthalene (3707 mg, 17.3 mmol) were dissolved in24.3 mL of trimethylphosphate. The solution was cooled to 0° C. Compound(5) (1.21 mL, 12.98 mmol) was added, and the mixture was stirred wellmaintaining the temperature at 0° C. for 5 hours. The reaction wasquenched by addition of 120 mL of tetraethyl-ammonium bromide solution(1M) and extracted with CH₂Cl₂ (2×80 mL). Purification was done bypreparative HPLC (Stationary phase: RP XBridge Prep C18 OBD-10 μm,30×150 mm, mobile phase: 0.25% NH₄HCO₃ solution in water, CH₃CN),yielding two fractions. The purest fraction was dissolved in water (15mL) and passed through a manually packed Dowex (H⁺) column by elutionwith water. The end of the elution was determined by checking UVabsorbance of eluting fractions. Combined fractions were frozen at −78°C. and lyophilized. Compound (9) was obtained as a white fluffy solid(303 mg, (0.86 mmol, 20% yield), which was used immediately in thefollowing reaction.

Step 2: Preparation of Compound (VI)

Compound (9) (303 mg, 0.86 mmol) was dissolved in 8 mL water and to thissolution was added N,N′-Dicyclohexyl-4-morpholine carboxamidine (253.8mg, 0.86 mmol) dissolved in pyridine (8.4 mL). The mixture was kept for5 minutes and then evaporated to dryness, dried overnight in vacuoovernight at 37° C. The residue was dissolved in pyridine (80 mL). Thissolution was added dropwise to vigorously stirred DCC (892.6 mg, 4.326mmol) in pyridine (80 mL) at reflux temperature. The solution was keptrefluxing for 1.5 h during which some turbidity was observed in thesolution. The reaction mixture was cooled and evaporated to dryness.Diethylether (50 mL) and water (50 mL) were added to the solid residue.N′N-dicyclohexylurea was filtered off, and the aqueous fraction waspurified by preparative HPLC (Stationary phase: RP XBridge Prep C18OBD-10 μm, 30×150 mm, mobile phase: 0.25% NH₄HCO₃ solution in water,CH₃CN), yielding a white solid which was dried overnight in vacuo at 38°C. (185 mg, 0.56 mmol, 65% yield). LC-MS: (M+H)+: 333.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 2.44-2.59 (m, 2H) signal falls underDMSO signal, 3.51 (td, J=9.90, 5.50 Hz, 1H), 3.95-4.11 (m, 2H), 4.16 (d,J=10.34 Hz, 1H), 4.25-4.40 (m, 2H), 5.65 (d, J=8.14 Hz, 1H), 5.93 (br.s., 1H), 7.46 (d, J=7.92 Hz, 1H), 2H's not observed

Biological Examples Replicon Assays

The compounds of formula I were examined for activity in the inhibitionof HCV-RNA replication in a cellular assay. The assay was used todemonstrate that the compounds of formula I inhibited a HCV functionalcellular replicating cell line, also known as HCV replicons. Thecellular assay was based on a bicistronic expression construct, asdescribed by Lohmann et al. (1999) Science vol. 285 pp. 110-113 withmodifications described by Krieger et al. (2001) Journal of Virology 75:4614-4624, in a multi-target screening strategy.

Replicon Assay (A)

In essence, the method was as follows. The assay utilized the stablytransfected cell line Huh-7 luc/neo (hereafter referred to as Huh-Luc).This cell line harbors an RNA encoding a bicistronic expressionconstruct comprising the wild type NS3-NS5B regions of HCV type 1btranslated from an internal ribosome entry site (IRES) fromencephalomyocarditis virus (EMCV), preceded by a reporter portion(FfL-luciferase), and a selectable marker portion (neo^(R), neomycinephosphotransferase). The construct is bordered by 5′ and 3′ NTRs(non-translated regions) from HCV genotype 1b. Continued culture of thereplicon cells in the presence of G418 (neo^(R)) is dependent on thereplication of the HCV-RNA. The stably transfected replicon cells thatexpress HCV-RNA, which replicates autonomously and to high levels,encoding inter alia luciferase, were used for screening the antiviralcompounds.

The replicon cells were plated in 384-well plates in the presence of thetest and control compounds which were added in various concentrations.Following an incubation of three days, HCV replication was measured byassaying luciferase activity (using standard luciferase assay substratesand reagents and a Perkin Elmer ViewLux™ ultraHTS microplate imager).Replicon cells in the control cultures have high luciferase expressionin the absence of any inhibitor. The inhibitory activity of the compoundon luciferase activity was monitored on the Huh-Luc cells, enabling adose-response curve for each test compound. EC₅₀ values were thencalculated, which value represents the amount of the compound requiredto decrease the level of detected luciferase activity by 50%, or morespecifically, the ability of the genetically linked HCV replicon RNA toreplicate.

Results (A)

Table 1 shows the replicon results (EC₅₀, replicon) and cytotoxicityresults (CC₅₀ (μM) (Huh-7)) obtained for the compound of the examplesgiven above.

TABLE 1 Compound EC₅₀ (μM) CC₅₀ (μM) number (HCV) (Huh-7) 8a 0.13 (n =4) >100

Replicon Assays (B)

Further replicon assays were performed with compound 8a of which theprotocols and results are disclosed below.

Assay 1

The anti-HCV activity of compound 8a was tested in cell culture withreplicon cells generated using reagents from the Bartenschlagerlaboratory (the HCV 1b bicistronic subgenomic luciferase reporterreplicon clone ET). The protocol included a 3-day incubation of 2500replicon cells in a 384-well format in a nine-point 1:4 dilution seriesof the compound. Dose response curves were generated based on thefirefly luciferase read-out. In a variation of this assay, a 3 dayincubation of 3000 cells in a 96-well format in a nine-point dilutionseries was followed by qRT-PCR Taqman detection of HCV genome, andnormalized to the cellular transcript, RPL 13 (of the ribosomal subunitRPL 13 gene) as a control for compound inhibition of cellulartranscription.

Assay 2

The anti-HCV activity of compound 8a was tested in cell culture withreplicon cells generated using reagents from the Bartenschlagerlaboratory (the HCV 1b bicistronic subgenomic luciferase reporterreplicon clone ET or Huh-Luc-Neo). The protocol included a 3-dayincubation of 2×10⁴ replicon cells in a 96-well format in a six-point1:5 dilution series of the compound. Dose response curves were generatedbased on the luciferase read-out.

Assay 3

The anti-HCV activity of compound 8a was tested in cell culture withreplicon cells generated using reagents from the Bartenschlagerlaboratory (the HCV 1b bicistronic subgenomic luciferase reporterreplicon clone ET or Huh-Luc-Neo). The protocol included either a 3-dayincubation of 8×10³ cells or 2×10⁴ cells in a 96-well format in aneight-point 1:5 dilution series of the compound. Dose response curveswere generated based on the luciferase read-out.

Results

Table 2 shows the average replicon results (EC₅₀, replicon) obtained forcompound 8a following assays as given above.

TABLE 2 Assay Average EC₅₀ value (8a): 1  57 μM (n = 8) 2  17.5 μM (n =4) 3 >100 μM (n = 1)

Primary Human Hepatocyte In Vitro Assay

The anti-HCV activity of compound 8a was determined in an in vitroprimary human hepatocyte assay. Protocols and results are disclosedbelow.

Protocol Hepatocyte Isolation and Culture

Primary human hepatocytes (PHH) were prepared from patients undergoingpartial hepatectomy for metastases or benign tumors. Fresh humanhepatocytes were isolated from encapsulated liver fragments using amodification of the two-step collagenase digestion method. Briefly,encapsulated liver tissue was placed in a custom-made perfusionapparatus and hepatic vessels were cannulated with tubing attachedmultichannel manifold. The liver fragment was initially perfused for 20min with a prewarmed (37° C.) calcium-free buffer supplemented withethylene glycol tetraacetic acid (EGTA) followed by perfusion with aprewarmed (37° C.) buffer containing calcium (CaCl₂), H₂O₂) andcollagenase 0.05% for 10 min. Then, liver fragment was gently shaken tofree liver cells in Hepatocyte Wash Medium. Cellular suspension wasfiltered through a gauze-lined funnel. Cells were centrifuged at lowspeed centrifugation. The supernatant, containing damaged or deadhepatocytes, non parenchymal cells and debris was removed and pelletedhepatocytes were re-suspended in Hepatocyte Wash Medium. Viability andcell concentration were determined by trypan blue exclusion test.

Cells were resuspended in complete hepatocyte medium consisting ofWilliam's medium (Invitrogen) supplemented with 100 IU/L insulin (NovoNordisk, France), and 10% heat inactivated fetal calf serum (Biowest,France), and seeded at a density 1,8×106 viable cells onto 6 well platesthat had been precoated with a type I collagen from calf skin(Sigma-Aldrich, France) The medium was replaced 16-20 hours later withfresh complete hepatocyte medium supplemented with hydrocortisonehemisuccinate (SERB, Paris, France), and cells were left in this mediumuntil HCV inoculation. The cultures were maintained at 37° C. in ahumidified 5% CO2 atmosphere.

The PHHs were inoculated 3 days after seeding. JFH1-HCVcc stocks wereused to inoculate PHHs for 12 hours, at a multiplicity of infection(MOI) of 0.1 ffu per cell. After a 12-hours incubation at 37° C., theinoculum was removed, and monolayers were washed 3 times withphosphate-buffered saline and incubated in complete hepatocyte mediumcontaining 0.1% dimethylsufoxide as carrier control, 100 IU/ml ofIFNalpha as negative control or else increasing concentrations ofcompound 8a. The cultures then were maintained during 3 days.

Quantitation of HCV RNA

Total RNA was prepared from cultured cells or from filtered culturesupernatants using the RNeasy or Qiamp viral RNA minikit respectively(Qiagen SA, Courtaboeuf, France) according to the manufacturer'srecommendations. HCV RNA was quantified in cells and culturesupernatants using a strand-specific reverse real-time PCR techniquedescribed previously (Carriere M and al 2007):

Reverse transcription was performed using primers described previouslylocated in the 50 NCR region of HCV genome, tag-RC1(5′-GGCCGTCATGGTGGCGAATAAGTCTAGCCATGGCGTIAGTA-3′) and RC21(5′-CTCCCGGGGCACTCGCAAGC-3′) for the negative and positive strands,respectively. After a denaturation step performed at 70° C. for 8 min,the RNA template was incubated at 4° C. for 5 min in the presence of 200ng of tag-RC1 primer and 1.25 mM of each deoxynucleoside triphosphate(dNTP) (Promega, Charbonnieres, France) in a total volume of 12 μl.

Reverse transcription was carried out for 60 min at 60° C. in thepresence of 20 U RNaseOut™ (Invitrogen, Cergy Pontoise, France) and 7.5U Thermoscript™ reverse transcriptase (Invitrogen), in the bufferrecommended by the manufacturer. An additional treatment was applied byadding 1 μl (2 U) RNaseH (Invitrogen) for 20 min at 37° C.

The first round of nested PCR was performed with 2 μl of the cDNAobtained in a total volume of 50 μl, containing 3 U Taq polymerase(Promega), 0.5 mM dNTP, and 0.5 μM RC1 (5′-GTCTAGCCATGGCGTIAGTA-3′) andRC21 primers for positive-strand amplification, or Tag(5′-GGCCGTCATGGTGGCGAATAA-3′) and RC21 primers for negative strandamplification. The PCR protocol consisted of 18 cycles of denaturation(94° C. for 1 min), annealing (55° C. for 45 sec), and extension (72° C.for 2 min). The cDNA obtained was purified using the kit from Qiagen,according to the manufacturer's instructions.

The purified product was then subjected to real-time PCR. The reactionwas carried out using the LightCycler 480 SYBR Green I Master (2× con)Kit (Roche, Grenoble, France), with LC480 instruments and technology(Roche Diagnostics). PCR amplifications were performed in a total volumeof 10 μl, containing 5 μl of Sybrgreen I Master Mix (2×), and 25 ng ofthe 197R (5′-CTITCGCGACCCAACACTAC-3′) and 104(5′-AGAGCCATAGTGGTCTGCGG-3′) primers. The PCR protocol consisted of onestep of initial denaturation for 10 min at 94° C., followed by 40 cyclesof denaturation (95° C. for 15 sec), annealing (57° C. for 5 sec), andextension (72° C. for 8 sec).

The quantitation of 28Sr RNA by specific RT-PCR was used as an internalstandard to express the results of HCV positive or negative strands perμg of total hepatocyte RNA. Specific primers for 28 S rRNA were designedusing the Oligo6 software 5′-TGAAAATCCGGGGGAGAG-3′(nt2717-2735) and50-ACATrGTCCAACATGCCAG-30 (nt 2816-2797). Reverse transcription wasperformed using AMV reverse transcriptase (Promega), and the PCRprotocol consisted of one step of initial denaturation for 8 min at 95°C., followed by 40 cycles of denaturation (95° C. for 15 sec), annealing(54° C. for 5 sec), and extension (72° C. for 5 sec).

Results

Table 3 shows the anti-HCV activity of compound 8a as determined in thein vitro primary human hepatocyte assay described above. The numbers areexpressed as 10⁶ HCV RNA copies/μg of total RNA. Results of twoindependent experiments (Exp 1 and Exp 2) are given. The data perexperiment is the average of two measurements.

Table 3: Effect of compound 8a on positive strand HCV-RNA levels inprimary human hepatocytes (expressed as 10⁶ HCV RNA copies/μg of totalRNA).

TABLE 3 Exp. 1 Exp. 2 No HCV 0 0 HCV control 3.56 5.53 IFNα (100 IU/mL)1.48 1.59 8a (0.195 μM) 2.18 1.12 8a (0.78 μM) 2.25 1.3 8a (3.12 μM)1.09 0.94 8a (12.5 μM) 2.17 1.3 8a(50 μM) 0.94 1.33

In Vivo Efficacy Assay

The in vivo efficacy of compound 8a and CAS-1375074-52-4 was determinedin a humanized hepatocyte mouse model (PBX-mouse) as previouslydescribed in Inoue et. al (Hepatology. 2007 April; 45(4):921-8) andTenato et. al. (Am J Pathol 2004; 165-901-912) with the followingspecification: Test animals: HCV G1a-infected PXB-mice, male orfemale, >70% replacement index of human hepatocytes. Dosing wasperformed p.o for 7 days at doses indicated below wherein QD representsa single dose per day, BID represents two doses per day.

Efficacy of compound 8a was compared to CAS-1375074-52-4. Results areindicated in FIG. 1. The FIGURE shows the log drop HCV viral RNA afterdosing for a period of 7 days.

FIG. 1 clearly shows that a dosing of 100 mg/kg QD for CAS 1375074-52-4(indicated as *, n=4) does not result in a significant log drop in HCVviral RNA. This in strong contrast to each of the indicated doseregimens for compound 8a, were a clear log drop is observed for 100mg/kg QD (indicated as *, n=3), 200 mg/kg QD (indicated as ●, n=4), 50mg/kg BID (indicated as ▪, n=4). The most pronounced log drop effect inviral RNA is observed after a 7 day dosing of compound 8a at 100 mg/kgBID (indicated as ▴, n=4).

1-13. (canceled)
 14. A compound of formula